AbstractThe alpha-diketone diacetyl (2,3-butanedione) is used as butter flavorant in the microwave popcorn industry and suspected to cause a rare obstructive pulmonary disease (popcorn lung) in workers. E-cigarette smoking is also a potential route of exposure to diacetyl and the potentially less toxic alternative 2,3-pentanedione. In this case study we are developing new-approach methodologies (NAMs) to reduce the uncertainty of read-across predictions. Primary bronchial epithelial cells were exposed to the alpha-diketones diacetyl, 2,3-pentanedione and 2,3-hexanedione or the beta-diketone 2,4-pentanedione under air-liquid interface (ALI) conditions using the P.R.I.T.® ExpoCube® device. This unique exposure device provides a highly efficient exposure situation by preventing contact between the test compound and the culture medium. Primary human bronchial epithelial cells (PBECs) from tumor-free lung tissues from four donors were differentiated into airway epithelium at ALI conditions. Test atmospheres were generated by evaporation of the volatile test compounds and diluted in clean air. FT-IR spectroscopy enabled online analysis of the exposure concentration. PBECs were exposed for 1h once or repeatedly on three consecutive days. Cellular viability was measured by LDH-leakage and monolayer integrity by measuring the transepithelial electrical resistance (TEER) 24h after the final exposure. Exposure concentrations ranged from 100 to 1840 ppm (diacetyl) and from 50 to 5000 ppm (other diketone analogues). Lowest observed adverse effect levels (LOAELs) were lower after repeated exposure compared to the single exposure protocol. NAM read outs correlated with alpha- and beta-diketone specific toxicity since 2,4-pentanedione displayed significantly lower cytotoxic effects in comparison to its analogues. In conclusion, in vitro testing of volatile gases enabled ranking of test compounds with regard to inhalation toxicity. Moreover, further comprehensive evaluations may adequately predict specific lung toxicity of structurally related compounds. Acknowledgement: This project received funding from the European Union’s Horizon 2020 research and innovation programme (grant agreement No 681002).